Creatine kinase

Dynabeads^® MyOne™ microbeads (average diameter of 1 μm) and FluidMAG-nanoparicles (average diameter of 50 nm) were purchased from Invitrogen (Carlsbad, CA, USA) and Chemicell (Berlin, Germany), respectively. RNase H was from Takara Bio Inc (Otsu, Japan). Nucleotide triphosphates, creatine phosphate (CP), and creatine kinase (CK) were purchased from Roche Applied Science (Indianapolis, IN, USA). All other chemical reagents were purchased from Sigma (St Louis, MO, USA) and used without further purification. The S12 extract was prepared from the strain BL21-Star^TM (DE3) (Invitrogen) following previously described procedures15 (link).

Freshly prepared egg extracts were supplemented with 0.005 mg/ml creatine kinase (Roche, 10127566001), 0.375 mM creatine phosphate (Roche, 10621714001), 0.05 mM ATP (Roche, 10519979001), 0.005 mM EGTA, 1 mM MgCl₂. In some experiments, delta-geminin was added (final concentration 1.5 μg/ml) to egg extract to inhibit DNA replication. Permeabilized sperm cells were added to a final concentration of 1000 nuclei/μl of extract and incubated at RT for 2 h with gentle tapping every 10 min. The reaction was stopped by adding 10 volumes of ice-cold Egg Lysis Buffer—Chromatin Isolation Buffer (ELB–CIB, 10 mM Hepes pH 7.8, 250 mM sucrose, 2.5 mM MgCl₂, 50 mM KCl, 1 mM DTT, 1 mM EDTA, 1 mM spermidine, 1 mM spermine, 0.1% Triton X-100, 10 mM sodium butyrate, 1× EDTA-free PI Cocktail (Roche, 05056489001)) following Wang et al.^{42 (link)}, with slight modifications. Chromatin was isolated via centrifugation at 4000 rpm for 5 min through a 0.3 mL sucrose cushion of ELB–CIB with 0.5 M sucrose underlayered in the tube. The pellet was washed once with ELB–CIB plus 250 mM KCl. Samples were aliquoted (0.5–1 million of cells per tube) and flash frozen in liquid nitrogen.

All chemicals were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise specified. Creatine kinase was purchased from Roche. Firefly luciferase and FITC-casein were purchased from Sigma Aldrich. Quantilum recombinant luciferase was purchased from Promega (Madison, WI). Hsp70 and Hsp40 were purchased from Enzo Life Sciences (Farmingdale, NY).

Transcription reactions were conducted with ∼5-20 ng/μl purified PCR product, in 40 mM HEPES pH 7.4, 6 mM MgCl₂, 20 mM spermidine (Sigma), 10 mM DTT, 0.5 mM ATP, 0.5 mM UTP, 0.5 mM CTP, 0.1 mM GTP (Roche), 0.5 mM CAP (NEB), 0.4-0.8 U/μL rRNasin (Promega), and 0.4 U/μL SP6 polymerase (NEB) at 37°C for 60 min (Sharma et al., 2010 (link)). In vitro translation reactions in a homemade rabbit reticulocyte (RRL) system containing 1/20 volume of transcription reaction, 0.5 μCi/μL ³⁵S-methionine (Perkin Elmer EasyTag), nuclease-treated crude rabbit reticulocyte (Green Hectares), 20 mM HEPES, 10 mM KOH, 40 μg/mL creatine kinase (Roche), 20 μg/mL pig liver tRNA, 12 mM creatine phosphate (Roche), 1 mM ATP (Roche), 1 mM GTP (Roche), 50 mM KOAc, 2 mM MgCl₂, 1 mM glutathione, 0.3 mM spermidine, and 40 μM of each amino acid except for methionine (Sigma), were at 32°C for 25 min unless otherwise indicated (Shao et al., 2013 (link), Sharma et al., 2010 (link)).

APC/C was immunopurified from 100 μL X. laevis egg extract with 2 μg anti-Cdc27 antibody (Santa Cruz, clone AF3.1) bound to 5 μL Protein-A Sepharose (Sigma), and ubiquitination reactions were performed as described^{18 (link),75 (link)}. Alternatively, Strep-tagged APC and its activator His-Cdh1 were purified from insect cells infected with baculovirus-based expression vectors^{76 (link)}. In the latter case, 0.015 μM Strep-APC and 0.3 μM His-Cdh1 were first preincubated on ice in 1X TBS (25 mM Tris-HCl [pH 7.5], 50 mM NaCl) for 30 min to activate the APC. The conjugation mixture contained 0.3 μM MBP-E1, 1.5 μM His-UbcH10, 0.13 μM His-Ube2S, 100 μM ubiquitin, 5 μM HA-tagged N-terminus fragment of cyclin B1 (HA-NCB1), and the activated APC/C. A 100 μL reaction was typically performed in 1X reaction buffer (25 mM Tris-HCl [pH 7.5], 50 mM NaCl, 10 mM MgCl₂) in the presence of ATP regeneration system (3.5 U/mL creatine kinase [Roche], 3.8 mM creatine phosphate disodium [Roche], 0.5 mM ATP disodium salt, 0.5 mM MgCl₂) for 5 h at 25 °C. Glycerol was added to the ubiquitinated NCB1 conjugates (HA-Ub_n-NCB1) to a final concentration of 10% (v/v) for storage at −80 °C.